NGF (Nerve Growth Factor) was the first regulator of neuron growth to be discovered and confirmed, and the best described neurotrophic factor. [Ho J L, He S, Hu A et al., J Exp Med, 1995, 181 (4):1493-1505]. NGF plays important roles in the stages of the proliferation and phenotypic differentiation of neural stem cells, the development of neurons, the growth of axons, the synthesis of neurotransmitters and cells' apoptosis, etc. [Sayada C, Denamur E, Elion J et al., Gene, 1992, 120 (1):129-130]. NGF regulates the differentiation and maturation of sympathetic and sensory neurons, is involved in supporting the normal function of the adult sympathetic neuron, and nutritionally supports the adult sensory neurons [Zhang D, Yang X, Berry J, et al., J In2fect Dis, 1997, 176 (4): 1035-1040]. The developments and differentiations of cholinergic neurons of basal forebrain and the cholinergic interneurons of striatum in the central nervous system are also regulated by NGF [Pal S, Barnhart K M, Wei Q, et al., Vaccine, 1999, 17 (5):459-465]. Because of the importance of NGF's physiologic activity, it is demonstrated that NGF is of very important clinical application values.
Human NGF consists of three types of peptide chains: α, β and γ, which are bound together by non-covalent bonds in the form of α2β2γ2. It is further demonstrated that the β subunit possesses the whole bioactivity of the NGF, wherein the β subunit is a dimmer consisting of two 118-amino-acid chains bonded by non-covalent bonds. There are three disulfide bonds in each monomer; and the correct formation of the three disulfide bonds (Cys58-Cys108, Cys68-Cys110, Cys15-Cys80) is the critical base for the protein folding, and then affects the bioactivity of NGF-β. Because of the vanishing concentration and content of human NGF-β in the adult human body and the unavailability of human tissue, it cannot be produced in large quantities via extraction from human tissues. And, the NGF-β preparation produced from the general bacteria (For example, E. coli) and yeast expression systems via recombinant technologies exhibits less bioactivity than the NGF-β extracted from animal organs. This is because there are not natural modification and dimerization in the these two systems [De Bernardez Clark E, Schwarz E, Rudolph, R et al., 1999; 309:217-36. Ikemura H, Takagi H, Inouye M, et al., J Biol Chem. 1987 Jun. 5; 262(16):7859-64. Nishizawa M, Ozawa F, Higashizaki T, et al., Appl. Microbiol Biotechnol. 1993 February; 38(5):624-30.]. Theoretically, there would be greater advantage to use mammal cells to express and prepare human NGF-β; however their low expression level and high cost tend to hamper large-scale production. [C. ANTHONY ALTAR, Louis E. BURTONt, GREGORY L. Proc. Natl. Acad. Sci. USA Vol. 88, pp. 281-285, January 1991)]. Moreover, the bioactivity of human NGF-β prepared by the non-human mammal cell system is incomparable to the NGF extracted from humans. One clinical trial based on the recombinant human NGF for diabetic neuropathy developed by Genentech, Inc. failed in Phase III because of poor therapeutic effect [Apfel SC. Int. Rev. Neurobiol 50: 393-413, 2002]. As of this writing, no recombinant human NGF has been approved as drugs on the market, though various strategies and efforts have been continually proposed based on recombinant technology in different expression systems such as bacteria, yeasts, CHO cells and insect cells.
The content of NGF in the submandibular gland is higher than in all remaining tissues or organs of adult male mouse. Moreover, mice propagate rapidly and are easy to be bred in large scale with compliance to industrial requirement. Thus, mice are supposed to be an ideal source for extracting and purifying NGF. The NGF-β with best biological activity and unlimited source on the market is extracted from mice submandibular gland. However, compared with human NGF, the therapeutic efficacy of mouse NGF on rodent experimental model of allergic myeloencephalitis and Parkinson's disease is not as good as that of human NGF. Furthermore, mouse NGF is more immunogenic to human than human nerve growth factor theoretically. And the stability and safety of human NGF maybe better than that of mouse NGF when applied in human.
There are a number of human genes are homologous to the mouse's, and the mouse is easy to feed because of the smaller size and relative shorter life cycle, thus mouse is one of the best model animal to investigate the gene function of mammal animals and human diseases. During the past several years, many kinds of mice have been genetic engineered to express heterogenous proteins through the gene targeting technology. The biological function of NGF and the interaction of NGF with its receptor are always attracting a lot interest in the field of neurobiology and developmental biology. Crowley et al used knock-out technology to destroy the mouse NGF gene, and investigated the influence of NGF on the development of neurons (Crowley C, Spencer S D, Nishimura M C, et. al, Cell. 1994 Mar. 25; 76(6):1001-11). It is demonstrated that the development and survival of sympathetic and sensory neuron depend strictly on NGF, and that this dependence cannot be compensated by other neurotrophins. Moreover, Smeyne et al. knocked out the cellular high-affinity receptor TrkA of NGF to investigate the function of TrkA in the development of neurons (Smeyne R J, Klein R, Schnapp A, et al. Nature. 1994 Mar. 17; 368(6468):193-4.). However, if all of the NGF gene or its receptor TrkA genes are knocked out, then the mouse will either die during its early embryonic development or be born with low vitality because of the depletion of NGF's physiological activity. This may be also one of the reason why there is no research to apply the knock-out strategy to study the specific interaction between NGF and its receptor. The mutant NGF obtained in vitro provides a good basis to investigate the interaction between NGF and its receptor. For example, the mutant NGF which binds the TrkA only (rather than the low affinity receptor P75) may be used to investigate the function of P75, which make the investigation more targeted, pertinent and specific (Horton A, Laramee G, Wyatt S, et al., Mol Cell Neurosci. 1997; 10(3-4):162-72. Ryden M, Hempstead B, Ibanez C F, et al., J Biol Chem. 1997 Jun. 27; 272(26):16322-8). But the in vitro gene mutant cannot reveal the effect of mutations during the individual development in the complex in vivo condition. Thus, it remains to be elucidated how to modify the NGF gene of animal to stably express mutein of NGF of said animal and study the function of NGF mutant and its receptor in the level of whole animal.